“Ca2+-independent Phosphatase Activity of the Plasma Membrane Ca2+ Pump”

DÉBORA E. RINALDI; HUGO P. ADAMO

Wood Hole, Massachusetts. EEUU.

Congreso; FIFTY- NINTH ANNUAL MEETING OF THE SOCIETY OF GENERAL PHYSIOLOGISTS.; 2005

50. CA2+-INDEPENDENT PHOSPHATASE ACTIVITY OF THE PLASMA MEMBRANE CA2+ PUMP. DÈBORA E. RINALDI and HUGO P. ADAMO, IQUIFIB-Facultad de Farmacia y Bioquímica (UBA-CONICET), Buenos Aires, Argentina (Sponsor: David Gadsby) The plasma membrane Ca2+ pump (PMCA), like the Na+-K+ ATPase and SERCA, is able to hydrolyze p-nitrophenylphosphate (pNPP). However, pNPP is ineffective in supporting the active transport of Ca2+ by the PMCA. We have reexamined the role of Ca2+, ATP, and calmodulin and acidic lipids in the phosphatase activity of the PMCA. In the absence of Ca2+, ATP, and calmodulin, the purified PMCA reconstituted in phosphatidylcholine liposomes was able to hydrolyze pNPP at a rate of 0.2 µmol/mg/min. The addition of 1 µM Ca2+ and 200 nM calmodulin increased the pNPPase fourfold. Reconstitution in liposomes containing phosphatidylserine eliminated the requirement of Ca2+-calmodulin for maximum phosphatase activity. Indeed Ca2+ inhibited this activity with an apparent affinity similar to that of the Ca2+ transport site. By homology with the SERCA pump, the Ca2+ binding site of the PMCA would be formed by main chain carbonyls of Val418, Ala 419, and Val421 and side chain carbonyls of Asn879, Asp883, and Glu423. A mutant of the human isoform 4xb in which Asn879 was replaced by Asp was expressed in Saccharomyces cerevisiae, solubilized by the detergent C12E10 and purified by calmodulin chromatography. The mutant reconstituted in liposomes containing phosphatidylserine had no measurable Ca2+-ATPase activity but it was capable of hydrolyzing pNPP. Furthermore, the pNPPase activity of Asn879Asp was resistant to Ca2+ inhibition. Altogether the results suggest that (a) the phosphatase activity of the PMCA does not require Ca2+, and thus the structural arrangement of the cytosolic domains should be similar to that of the E2 conformation; (b) changing Asn879 to Asp disrupts the Ca2+ binding site, thereby stabilizing the enzyme in an E2-like conformation; and (c) in E2, maximum phosphatase activity is observed when the pump is not inhibited by the COOH-terminal autoinhibitory domain. [Supported by grants of CONICET and UBA.]